Structural insights into fusidic acid resistance and sensitivity in EF-G

Fusidic acid (FA) is a steroid antibiotic commonly used against Gram positive bacterial infections. It inhibits protein synthesis by stalling elongation factor G (EF-G) on the ribosome after translocation. A significant number of the mutations conferring strong FA resistance have been mapped at the interfaces between domains G, III and V of EF-G. However, direct information on how such mutations affect the structure has hitherto not been available. Here we present the crystal structures of two mutants of Thermus thermophilus EF-G, G16V and T84A, which exhibit FA hypersensitivity and resistance in vitro, respectively. These mutants also have higher and lower affinity for GTP respectively than wild-type EF-G. The mutations cause significant conformational changes in the switch II loop that have opposite effects on the position of a key residue, Phe90, which undergoes large conformational changes. This correlates with the importance of Phe90 in FA sensitivity reported in previous studies. These structures substantiate the importance of the domain G/domain III/domain V interfaces as a key component of the FA binding site. The mutations also cause subtle changes in the environment of the "P-loop lysine", Lys25. This led us to examine the conformation of the equivalent residue in all structures of translational GTPases, which revealed that EF-G and eEF2 form a group separate from the others and suggested that the role of Lys25 may be different in the two groups.     

Lactate induces tumour necrosis factor-alpha, interleukin-6 and interleukin-1beta release in microglial- and astroglial-enriched primary cultures

Hyperammonaemia has deleterious effects on the CNS in patients with liver dysfunction. Cellular mechanisms underlying the effects of hyperammonaemia are largely unknown, although astrocytes have been the main target of interest. This study investigated how treatment with NH4Cl and lactate, which increase in the brain as a consequence of hyperammonaemia, affects cells in primary rat cultures enriched in either astrocytes or microglia. Morphological changes were studied over time using light microscopy. Release of the proinflammatory cytokines tumour necrosis factor-alpha (TNF-alpha), interleukin (IL)-6 and IL-1beta was measured using ELISA. NH4Cl was found to induce vacuole formation in both culture systems. Lactate treatment altered astrocytic appearance, resulting in increased space between individual cells. Microglia adopted a round morphology with either NH4Cl or lactate treatment. Lactate, but not NH4Cl, induced release of TNF-alpha and IL-6 in both astroglial- and microglial-enriched cultures, while IL-1beta was released only in microglial cultures. Cytokine release was higher in the microglial- than in the astroglial-enriched cultures. Additionally, the astroglial-enriched cultures containing approximately 10% microglial cells released more cytokines than cultures containing about 5% microglial cells. Taken together, our data suggest that most TNF-alpha, IL-6 and IL-1beta release comes from microglia. Thus, microglia could play an important role in the pathological process of hyperammonaemia.     

The evolution of host use and unusual reproductive strategies in Achrysocharoides parasitoid wasps

We studied host selection and exploitation, two crucial aspects of parasite ecology, in Achrysocharoides parasitoid wasps, which show remarkable host specificity and unusual offspring sex allocation. We estimated a molecular phylogeny of 15 Achrysocharoides species and compared this with host (plant and insect) phylogenies. This tri-trophic phylogenetic comparison provides no evidence for cospeciation, but parasitoids do show phylogenetic conservation of the use of plant genera. Patterns of sequence divergence also suggest that the parasitoids radiated more recently (or evolved much faster) than their insect hosts. Three main categories of brood production occur in parasitoids: (1) solitary offspring, (2) mixed sex broods and (3) separate (split) sex broods. Split sex broods are very rare and virtually restricted to Achrysocharoides, while the other types occur very widely. Our phylogeny suggests that split sex broods have evolved twice and provides evidence for a transition from solitary to mixed sex broods, via split sex broods, as predicted by theory.     

Linkage mapping reveals sex-dimorphic map distances in a passerine bird

Linkage maps are lacking for many highly influential model organisms in evolutionary research, including all passerine birds. Consequently, their full potential as research models is severely hampered. Here, we provide a partial linkage map and give novel estimates of sex-specific recombination rates in a passerine bird, the great reed warbler (Acrocephalus arundinaceus). Linkage analysis of genotypic data at 51 autosomal microsatellites and seven markers on the Z-chromosome (one of the sex chromosomes) from an extended pedigree resulted in 12 linkage groups with 2-8 loci. A striking feature of the map was the pronounced sex-dimorphism: males had a substantially lower recombination rate than females, which resulted in a suppressed autosomal map in males (sum of linkage groups: 110.2 cM) compared to females (237.2 cM; female/male map ratio: 2.15). The sex-specific recombination rates will facilitate the building of a denser linkage map and cast light on hypotheses about sex-specific recombination rates.     

Specific and sensitive detection of the conifer pathogen <Emphasis Type="Italic">Gremmeniella abietina</Emphasis> by nested PCR

Background  

Gremmeniella abietina (Lagerb.) Morelet is an ascomycete fungus that causes stem canker and shoot dieback in many conifer species. The fungus is widespread and causes severe damage to forest plantations in Europe, North America and Asia. To facilitate early diagnosis and improve measures to control the spread of the disease, rapid, specific and sensitive detection methods for G. abietina in conifer hosts are needed.     

A nine-transmembrane domain topology for presenilin 1

Presenilin (PS) provides the catalytic core of the gamma-secretase complex. Gamma-secretase activity leads to generation of the amyloid beta-peptide, a key event implicated in the pathogenesis of Alzheimer disease. PS has ten hydrophobic regions, which can all theoretically form membrane-spanning domains. Various topology models have been proposed, and the prevalent view holds that PS has an eight-transmembrane (TM) domain organization; however, the precise topology has not been unequivocally determined. Previous topological studies are based on non-functional truncated variants of PS proteins fused to reporter domains, or immunocytochemical staining. In this study, we used a more subtle N-linked glycosylation scanning approach, which allowed us to assess the topology of functional PS1 molecules. Glycosylation acceptor sequences were introduced into full-length human PS1, and the results showed that the first hydrophilic loop is oriented toward the lumen of the endoplasmic reticulum, whereas the N terminus and large hydrophilic loop are in the cytosol. Although this is in accordance with most current models, our data unexpectedly revealed that the C terminus localized to the luminal side of the endoplasmic reticulum. Additional studies on the glycosylation pattern after TM domain deletions, combined with computer-based TM protein topology predictions and biotinylation assays of different PS1 mutants, led us to conclude that PS1 has nine TM domains and that the C terminus locates to the lumen/extracellular space.     

Regulation of sterol 27-hydroxylase in human monocyte-derived macrophages: up-regulation by transforming growth factor beta1

Regulatory mechanisms for human CYP27A1 enzyme have not yet been fully investigated. Our approach was to add different hormones and cytokines to cultured human monocyte-derived macrophages, and assess the effects on the CYP27A1 by measuring the production of 27-hydroxylated cholesterol in the media. Of the different hormones and cytokines tested, only transforming growth factor beta1 (TGF-beta1) had a clear effect on CYP27A1. Further experiments showed a significant increase in 27-hydroxylated cholesterol products (27-hydroxycholesterol and 3beta-hydroxy-5-cholestenoic acid). A concomitant increase in CYP27A1 mRNA levels was also seen and this positive effect was confirmed using a human CYP27A1 luciferase reporter gene expressed in HepG2 cells. Experiments with progressive deletion/luciferase reporter gene constructs indicated that a TGF-beta1 responsive sequence might be localized in a region about 400 bp upstream of the CYP27A1 translation start. The possibility is discussed that induction of CYP27A1 by TGF-beta1 may be responsible for some of the anti-atherogenic properties of this cytokine.     

DOES LINKAGE DISEQUILIBRIUM GENERATE HETEROZYGOSITY‐FITNESS CORRELATIONS IN GREAT REED WARBLERS?

Heterozygosity-fitness correlations (HFCs) at noncoding genetic markers are commonly assumed to reflect fitness effects of heterozygosity at genomewide distributed genes in partially inbred populations. However, in populations with much linkage disequilibrium (LD), HFCs may arise also as a consequence of selection on fitness loci in the local chromosomal vicinity of the markers. Recent data suggest that relatively high levels of LD may prevail in many ecological situations. Consequently, LD may be an important factor, together with partial inbreeding, in causing HFCs in natural populations. In the present study, we evaluate whether LD can generate HFCs in a small and newly founded population of great reed warblers (Acrocephalus arundinaceus). For this purpose dyads of full siblings of which only one individual survived to adult age (i.e., returned to breed at the study area) were scored at 19 microsatellite loci, and at a gene region of hypothesized importance for survival, the major histocompatibility complex (MHC). By examining siblings, we controlled for variation in the inbreeding coefficient and thus excluded genome-wide fitness effects in our analyses. We found that recruited individuals had significantly higher multilocus heterozygosity (MLH), and mean d2 (a microsatellite-specific variable), than their nonrecruited siblings. There was a tendency for the survivors to have a more diverse MHC than the nonsurvivors. Single-locus analyses showed that the strength of the genotype-survival association was especially pronounced at four microsatellite loci. By using genotype data from the entire breeding population, we detected significant LD between five of 162 pairs of microsatellite loci after accounting for multiple tests. Our present finding of a significant within-family multilocus heterozygosity-survival association in a nonequilibrium population supports the view that LD generates HFCs in natural populations.     

Glucose and fatty acid metabolism in McA-RH7777 hepatoma cells vs. rat primary hepatocytes: responsiveness to nutrient availability

The overabundance of dietary fats and simple carbohydrates contributes significantly to obesity and metabolic disorders associated with obesity. The liver balances glucose and lipid distribution, and disruption of this balance plays a key role in these metabolic syndromes. We investigated (1) how hepatocytes balance glucose and fatty acid metabolism when one or both nutrients are supplied in abundance and (2) whether rat hepatoma cells (McA-RH7777) reflect nutrient partitioning in a similar manner as compared with primary hepatocytes. Increasing media palmitate concentration increased fatty acid uptake, triglyceride synthesis and beta-oxidation. However, hepatoma cells had a 2-fold higher fatty acid uptake and a 2-fold lower fatty acid oxidation as compared with primary hepatocytes. McA-RH7777 cells did not synthesize significant amounts of glycogen and preferentially metabolized the glucose into lipids or into oxidation. In primary hepatocytes, the glucose was mostly spared from oxidation and instead partitioned into both de novo glycogen and lipid synthesis. Overall, lipid production was rapidly induced in response to either glucose or fatty acid excess and this may be one of the earliest indicators of metabolic syndrome development associated with nutrient excess.     

Molecular evidence of a reed warbler × great reed warbler hybrid ( Acrocephalus scirpaceus × A. arundinaceus) in Belgium

We report on the second case of a reed warbler × great reed warbler hybrid (Acrocephalus scirpaceus and A. arundinaceus). The bird was captured during a standardised ringing session in Belgium in autumn 1999, and fell between the parental species in all measurements. Molecular analyses of two microsatellite loci verified that the bird was a female that had a reed warbler father and a great reed warbler mother.